A. Biological Activity of Coumarin Derivatives
Anticoagulant and antithrombotic activity of certain natural and synthetic coumarin derivatives is known. See, Murray et al., The Natural Coumarins, Wiley, New York, 1982. Certain coumarin derivatives are also reported as triplet sensitizers (see, Williams et al., Polym. Eng. Sci., 1983, 23, 1022); anti-HIV agents (Spino et al., Bioorg. Med. Chem. Lett., 1998, 8, 3475-78); lipid-lowering agents (Madhavan et al., Bioorg. Med. Chem. Lett., 2003, 13, 2547-51); antioxidants (Kontogiorgis et al., J. Enzyme Inhib. Med. Chem., 2003, 18, 63-69); inhibitors of lipid peroxidation and vasorelaxant agents (Hoult et al., Gen. Pharmac. 1996, 27, 713-22); anti-inflammatory agents (Khan et al., Indian J. Chem., 1993, 32, 817); and free radical scavengers (Mora et al., J. Biochem. Pharmacol., 1990, 40, 793-97). In addition, two naturally-occurring coumarins have been found to exhibit cytotoxicity across a selection of mammalian cancer cell lines (Reutrakul et al., Planta Med., 2003, 69, 1048-51).
Certain coumarin-3-carboxamides have been reported as inhibitors of proteases, including α-chymotrypsin (Pochet et al., Bioorg. Med. Chem. Lett., 2000, 8, 1489-501; Wouters et al., Bioorg. Med. Chem. Lett., 1990, 12, 1109-12; and Mor et al., Biochim. Biophys. Acta, 1990, 1038, 119-24) and human leukocyte elastase (HLE) (Doucet et al., J. Med. Chem., 1999, 42, 4161-71; Egan et al., Drug Metab. Rev., 1990, 22, 503-29; and Nicolaides et al., J. Heterocycl. Chem., 1996, 33, 967).
B. Cyclin Dependent Kinase (CDK) Inhibition
One of the most important and fundamental processes in biology is the division of cells mediated by the cell cycle. The cell cycle is regulated by a diverse set of cellular signals both within the cell and from external sources. A complex network of tumor promoting and suppressing gene products are key components of this cellular signaling process. Overexpression of the tumor promoting components or the subsequent loss of the tumor suppressing products may lead to unregulated cellular proliferation and the generation of tumors. CDKs serve to regulate the cell cycle. CDK complexes comprise a catalytic subunit (the kinase) and a regulatory subunit (the cyclin). Nine kinase subunits (CDK 1-9) have been identified along with several regulatory subunits (cyclins A-H).
CDKs are important targets for therapeutic intervention in various proliferative disorders including cancer. Each kinase associates with a specific regulatory partner and together make up the active catalytic moiety. Each transition of the cell cycle is regulated by a particular CDK complex. The coordinated activity of these kinases guides the individual cells through the replication process and ensures the vitality of each subsequent generation.
Overexpression of the cyclin regulatory proteins and subsequent kinase hyperactivity have been linked to several types of cancers (Jiang, Proc. Natl. Acad. Sci. USA 90:9026-9030, 1993; Wang, Nature 343:555-557, 1990). Endogenous CDK inhibitors (e.g., p16INK4 (an inhibitor of CDK4/D1), p21CIP1 (a general CDK inhibitor), and p27KIP1 (a specific CDK2/E inhibitor) have been shown to affect cellular proliferation (Kamb et al., Science 264:436-440, 1994; Beach, Nature 336:701-704, 1993). These inhibitors help to regulate the cell cycle through specific interactions with their corresponding CDK complexes. Cells deficient in these inhibitors are prone to unregulated growth and tumor formation. CDKs are also known to play a role in apoptosis.
New CDK inhibitors, particularly small molecule inhibitors, would be useful in the treatment of cell proliferative disorders such as cancer, familial adenomatosis polyposis, neuro-fibromatosis, psoriasis, fungal infections, endotoxic shock, transplantation rejection, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis. U.S. Pat. No. 6,114,365 discloses that CDK inhibitors are useful in the treatment of cancers that include carcinoma such as bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma. See, U.S. Pat. No. 6,114,365, the entire disclosure of which is incorporated herein by reference.
Cell cycle control is also implicated in viral replication. CDK9 is known to activate Tat, a nuclear transcriptional activator encoded by Human Immunodeficiency Virus (HIV). HIV type 1 (HIV-1) can infect quiescent cells. However, viral production is restricted to actively proliferating cells. The HIV-1 viral protein Tat acts to perturb the cell cycle thereby optimizing HIV-1 replication. Tat regulates the cell cycle by altering factors involved in proliferation and differentiation, and by associating with cyclin/CDK complexes.
Tat protein is a potent activator of HIV-1 transcription that functions at an early step in elongation. Tat acts to enhance the processivity of RNA polymerase II (RNAPII) complexes that would otherwise terminate transcription prematurely at random locations downstream of the viral RNA start site. The mechanism of Tat transactivation is unique in that the cis-acting transactivation response element (TAR) is a stable RNA stem-loop structure that forms at the 5′ end of nascent viral transcripts. Transcriptional activation by Tat through TAR requires proper folding of the RNA as well as specific bases in the bulge and apical loop of the TAR RNA hairpin structure. See, Cullen, Cell 73:417-420 (1993) and Jones et al., Ann. Rev. Biochem. 63:717-743 (1994) the entire disclosures of which are incorporated herein by reference.
The role for CDK9 in Tat transactivation has been shown in random drug screens for specific inhibitors of Tat, which yield novel compounds directed against the active site of CDK9 (Mancebo et al. (1997) Genes Dev 11:2633-2644). In addition a dominant negative mutant CDK9 protein has been shown to block Tat activity in vivo (Id.; Yang et al. (1997) Proc. Natl. Acad. Sc. USA 94:12331-12336). Thus, inhibitors of CDK9 represent a means to treat viral infection by inhibiting viral replication. Inhibition of CDK9 has also been shown to inhibit replication of other viruses including varicella-zoster virus and herpes simplex. See, Taylor et al., J. Virol., 78(6), page 2853-62 (2004), the entire disclosure of which is incorporated herein by reference.
Cancer and other proliferative disorders remain a major unmet medical need. Cancer treatments often comprise surgery, chemotherapeutic treatments, radiation treatment or combinations thereof. Chemotherapeutic treatments for most cancers only delay disease progression rather than providing a cure. Cancers often become refractory to chemotherapy via development of multidrug resistance. Particular cancers are inherently resistant to some classes of chemotherapeutic agents. See, DeVita et al, “Principles of Cancer Management: Chemotherapy” In: Cancer. Principles and Practice of Oncology, 5th edition, Lippincott-Raven, Philadelphia, New York (1977), pp. 333-347.
Viral infection represents another area of major unmet medical need. Viruses often develop resistance. Present therapies often demonstrate significant toxicity at therapeutic doses, and even then serve only to slow progression of the viral disorder.
Thus, there remains a need to develop new therapeutic agents. Oncoproteins in general, and signal transducing proteins, such as CDKs in particular, are likely to be more selective targets for chemotherapy because they represent a subclass of proteins whose activities are essential for cell proliferation, and because their activities are greatly amplified in proliferative diseases.